Maintenance of epithelial polarity is essential for proper function and homeostasis the kidney. This requires the targeted delivery of newly synthesized and recycled proteins to the apical and basolateral surfaces. Disregulation of protein delivery can lead to serious diseases such as hypertension, diabetes, and lysosomal storage diseases. The long term objective of my project is to examine the signals and mechanisms that regulate the apical delivery of secreted proteins. Recent studies from our lab and others have demonstrated that there are multiple routes to the apical surface of polarized renal cells. The aims of my proposal are: 1) to determine whether secreted proteins traverse endocytic compartments en route to the apical surface of polarized epithelial cells;and 2) to characterize post-Golgi compartments involved in biosynthetic delivery of apically-destined proteins. I will use the Madin-Darby canine kidney (MDCK) cell line to investigate polarized delivery of distinct classes of secreted apical proteins. MDCK cells are a valuable model because they emulate the structural and functional polarity of the kidney tubule. In aim 1 I will examine the biosynthetic route of two apically secreted proteins that are sorted via distinct mechanisms in MDCK cells using biochemical and live-cell imaging approaches. In aim 2 I will use a novel flow-cytometry approach to selectively collect post-Golgi carriers containing fluorescent protein-tagged versions of these markers and employ proteomic approaches to identify other protein components enriched in carriers that ferry secreted proteins. The results of my studies will provide new insights into the mechanisms that regulate sorting and trafficking of this important class of proteins. The disruption of normal kidney function can be related to diseases such as, hyper- and hypo-tension, and diabetes. The goal of our research is to study kidney cells and how they process and produce proteins that are critical for maintaining kidney health. Ultimately, our research will provide the basis for development of highly specific treatments for patients with kidney diseases.